The goal of this project is to systematically survey the evolution of pre-mRNA splicing in primates, and elucidate the molecular mechanisms that created species-specific exons and splicing patterns. Alternative splicing in higher eukaryotes generates an enormous regulatory and functional diversity from a limited repertoire of protein-coding genes. It also permits a gene to evolve a new spliced isoform, while still expressing the ancestral spliced isoform. Many genes have species-specific exons and splicing patterns that arose from either small-scale sequence changes that affected essential splicing signals, or large-scale insertions or deletions. However, despite the critical role of splicing during eukaryotic genome evolution, many questions regarding how splicing changes occurred and the evolutionary significance of such changes remain largely unexplored. We propose to combine genomic, computational, and molecular approaches to study splicing changes during primate and human evolution. The specific aims are: 1) To investigate the birth and evolution of new exons in primates, using genome alignments of vertebrate species, extensive exon-level transcriptome profiles of human genes generated by microarray and sequencing-based technologies, and molecular splicing analysis of new exons in humans and nonhuman primates. 2) To globally examine splicing differences between humans and nonhuman primates, by high-density exon junction array and RNA-seq profiling of a large panel of human and primate tissues. 3) To elucidate the mechanisms of splicing evolution in primates, via comparative analysis of splicing regulatory signals and minigene experiments. This project will improve the annotation of human and primate genomes, greatly expand the knowledge of new exons and splicing patterns that are unique to our species, and shed light on how eukaryotic genomes expand their functional repertoire via the evolution of splicing. The results of these studies will elucidate how the evolution of genomic sequences contributed to splicing differences among species. This will provide significant insight into the regulation of splicing, and how genetic variations disrupt splicing in human diseases. PUBLIC HEALTH RELEVANCE: Many human diseases are caused by aberrations in pre-mRNA splicing. This project will systematically survey the evolution of splicing in primates, and elucidate how splicing patterns change as a result of genome sequence evolution. These studies will provide significant insight into how splicing is regulated, and how genetic variations disrupt splicing in human diseases.